Microbiological production of polyesters

ABSTRACT

The invention relates to a microbiological process for the production of polyesters and utilizes bacteria of the Pseudomanas fluorescens rRNA branch according to the phylogenetic classification of De Vos and De Ley. These bacteria are cultured under aerobic fermentation conditions in a nutrient medium comprising an excess of at least one assimilarable acylic aliphatic hydrocarbon compound having 6-18 carbon atoms and a limiting quantity of at least one of other nutrients essential for growth to form poly-3-hydroxyalkanaoates.

This invention relates to a process for producing polyesters byaerobically culturing microorganisms, preferably with nutrientlimitation. More in particular, the invention relates to a process forproducing polyesters by culturing Pseudomonas bacteria under aerobicconditions in a nutrient medium preferably containing an excess of acarbon source and a limiting quantity of at least one of the othernutrients essential for growth, the carbon source comprising at leastone assimilable acyclic aliphatic hydrocarbon compound, and, if desired,recovering the biopolymer formed from the cells.

Such a process is known from European patent application EP-A-0 274 151.According to the process described therein Pseudomonas oleovoransbacteria are used which surprisingly proved capable of convertinghydrocarbon compounds with certain nutrient limitations into polymericproducts. The polymers formed were found to differ from the known PHB,i.e. poly(3-hydroxy-butyrate). It turned out that they were built upfrom units having the formula (1): --CO--CH₂ --CH [(CH₂)_(m) CH₃ ]--O--,and/or units having the formula (2): --CO--CH₂ --CH [(CH₂)_(m-1) CH═CH₂]--O--, in which m is an integer of 2-8. The composition of thebiopolymers formed by the bacteria [which will hereinafter be referredto as PHA, i.e. poly(3-hydroxy--alkanoate)] proved dependent on thenature of the hydrocarbon compound present in the medium. When, e.g.,the substrate used was n-decane, the polymer formed proved to consist of3-hydroxy-decanoate, 3-hydroxy-octanoate and 3-hydroxy-hexanoate units.On the other hand, when the substrate used was n-undecane, the polymerformed proved to consist of 3-hydroxy-undecanoate, 3-hydroxy-nonanoateand 3-hydroxy-heptanoate units. When the substrate used was anunsaturated hydrocarbon compound (1-olefins, such as 1-octene), thepolymer formed also comprised units of formula (2).

Surprisingly, it has now been found that bacterial species other thanPseudomonas oleovorans can be used as well, namely bacteria belonging tothe pseudomonads of the Pseudomonas fluorescens rRNA branch according tothe phylogenetic classification by de Vos and de Ley, Int. J. of Syst.Bacteriol. 33, 1983, 487-509. According to this classification,different Pseudomonas branches can be distinguished in ribosomal RNA byhomology, namely the rRNA branch of Pseudomonas solanacearum, the rRNAbranch of Pseudomonas acidovorans and the rRNA branch of Pseudomonasfluorescens. The bacteria belonging to the two first branches are foundto be PHB formers, while the bacteria belonging to the last-mentionedbranch have in con, non that on certain carbon sources (not on sugars,methanol or short fatty acids) they form no PHB but PHA's, especiallywith nutrient limitation (starvation).

PHB-forming pseudomonads of the rRNA branch of Pseudomonas solanacearumare P. solanacearum, P. cepacia, P. marginata, P. caryophili and P.lemoignei. PHB-forming pseudomonads of the rRNA branch of Pseudomonasacidovorans are P. acidovorans, P. delafieldii, P. testosteroni, P.facilis, P. palleronii and P. ftava. Pseudomonads of the rRNA branch ofPseudomonas fluorescens forming no PHB but PHA are P. fluorescens,biotype I (inter alia the prototype), P. fluorescens of biotype II, P.fluorescens of biotype III, P. fluorescens of biotype IV (such as P.lemonnieri), P. putida biotype A (which, inter alia, includes P.oleovorans), P. putida biotype B, P. aureofaciens, P. syringae, P.stutzeri, P. mendocina, P. chloraohis, P. cichorii, P.pseudoalcaligenes, P. alcaligenes and P. aeruginosa. The pseudomonads ofthe Pseudomonas fluorescens rRNA branch according to the phylogeneticclassification by de Vos and de Ley in Int. J. of Syst. Bacteriol. 33,1983, 487-509, correspond to the pseudomonads from group I according tothe determination in Bergey's Manual of Determinative Bacteriology.

When selecting the substrate, it must be considered that bacterialspecies of the Pseudomonas fluorescens rRNA branch other thanPseudomonas oleovorans are mostly incapable of metabolizing paraffins.The ability of P. oleovorans to metabolize paraffins is due toplasmid-encoded (OCT plasmid) enzymes which are involved in the firststeps of the paraffin oxidation. On the other hand, paraffin oxidationproducts, such as alkanols, alkanals, paraffin carboxylic acids andparaffin dicarboxylic acids, can often be metabolized by these otherbacterial species. Also, the length of the hydrocarbon compound servingas a substrate preferred by a selected bacterial species may bedifferent from the length preferred by P. oleovorans. While in case ofbacteria of the species P. oleovorans the best results are obtained byusing C₆ -C₁₂ paraffins and alkanols or C₆ -C₁₈ (un)saturated fattyacids as a substrate, it is better in case of P. aeruginosa bacteria touse C₁₂ -C₁₆ paraffins and alkanols or C₆ -C₁₀ paraffin dicarboxylicacids.

In the first place, the invention therefore provides a process forproducing polyesters by culturing bacteria of the Pseudomonasfluorescens rRNA branch according to the phylogenetic classification byde Vos and de Ley in Int. J. of Syst. Bacteriol 33, 1983, 487-509, withthe exception of Pseudomonas oleovorans, under aerobic conditions in anutrient medium comprising as a carbon source at least one assimilableacyclic aliphatic hydrocarbon compound, and, if desired, recovering thebiopolymer formed from the cells.

The invention particularly provides a process for producing polyestersby culturing bacteria of the Pseudomonas fluorescens rRNA branchaccording to the phylogenetic classification by de Vos and de Ley inInt. J. of Syst. Bacteriol. 33, 1983, 487-509, with the exception ofPseudomonas oleovorans, under aerobic conditions in a nutrient mediumcomprising an excess of a carbon source and a limiting quantity of atleast one of the other nutrients essential for growth, the carbon sourcecomprising at least one assimilable acyclic aliphatic hydrocarboncompound, and, if desired, recovering the biopolymer formed from thecells.

More in particular, the invention provides such a process, whichcomprises the use of bacteria selected from the group consisting of

Pseudomonas fluorescens, biotype I

Pseudomonas fluorescens, biotype II

Pseudomonas fluorescens, biotype III

Pseudomonas fluorescens, biotype IV

Pseudomonas putida biotype A (except P. oleovorans)

Pseudomonas putida biotype B

Pseudomonas aureofaciens

Pseudomonas syringae

Pseudomonas stutzeri

Pseudomonas mendocina

Pseudomonas chloraphis

Pseudomonas cichorii

Pseudomonas pseudoalcaligenes

Pseudomonas alcaligenes

Pseudomonas aeruginosa.

In the process according to the invention a saturated or unsaturatedparaffin or paraffin oxidation product having 6 or more carbon atoms isgenerally used as the assimilable acyclic aliphatic hydrocarboncompound. More in particular, the substrate used is a saturated orunsaturated paraffin or paraffin oxidation product having 6-24 carbonatoms, preferably 6-18 carbon atoms. The term "paraffin oxidationproduct" as used herein particularly means alkanols, alkanals, alkanoicacids and paraffin dicarboxylic acids occurring as intermediates in thenatural paraffin decomposition. Of course, steps must be taken, ifnecessary, to avoid any toxic effect of such substances. For thisreason, when the substrates used are, e.g., alkanols or alkanals, aninert auxiliary phase, such as dibutyl phthalate, will usually be addedto the medium for the purpose of dilution. Preferably, however, thesubstrate used is a paraffin carboxylic acid having 6-18, preferably12-16 carbon atoms, or a paraffin dicarboxylic acid having 6-18,preferably 6-12 carbon atoms. If desired, there is also used a reactant,such as Brij 58, to keep these substrates in solution.

The composition of the polymers obtained by the process according to theinvention depends on the substrate used. When a substrate having an evennumber of carbon atoms is used, the units constituting the polymer alsohave an even number of carbon atoms. Similarly, the polymer units havean odd number of carbon atoms when a substrate having an odd number ofcarbon atoms is used. The smallest polymer units have 6 and 7 carbonatoms, respectively. However, also when substrates having more than 12carbon atoms are used, the polymers formed essentially do not containunits having more than 12 carbon atoms. In practice, the units having 8and 9 carbon atoms, respectively, are always found to be predominant.

In order to stimulate the bacteria to form PHA, a nutrient limitation ispreferably used, i.e. in practice, the aerobic cultivation is carriedout with a nitrogen, phosphorus, sulfur or magnesium limitation,preferably on a medium, such as an E2-medium, with a nitrogenlimitation. The growth and polymer forming conditions are essentially asdescribed in European patent application EP-A-0 274 151. According tothese commonly used conditions the aerobic cultivation is carried out(fed batchwise or continuously) at pH 5-9, preferably 7, at atemperature below 37° C., preferably about 30° C., and with adequateagitation at a dissolved oxygen tension above 30%, preferably about 50%or more saturation with air. If desired, a system with two liquid phasesis used, one of which is an aqueous phase containing the water-solublenutrients and the bacteria, and the other of which is an apolar phasecontaining the hydrocarbon compound(s) serving as a substrate. However,when more polar substrates are used, such as the mono- and dicarboxylicacids, the system preferred will be a one-phase system using suitablesurfactants.

In practice, the procedure will be commonly such that the aerobiccultivation with nutrient limitation is preceded by an exponentialgrowth phase without nutrient limitations until a cell density of atleast 2 g/l is reached. It is also possible, however, to have aco-metabolic process taken place, in which a preferably inexpensivecarbon source, such as glucose, sucrose, molasses etc., is used forgrowth (i.e. for multiplying cell mass) and a paraffin or paraffinoxidation product is used as a substrate for the desired polymerformation.

The stationary phase, in which the biopolymer inclusions are formed, ispreferably not continued too long, because, after reaching a maximum,the polymer content is again decreased. Preferably, therefore, thebiopolymer containing cells formed in the stationary phase with nutrientlimitation are harvested before a significant decrease of the biopolymercontent of the cells has taken place.

However, it has been established by way of experiment that PHA's canalso be formed under non-limiting conditions. Thus, bacteria of thestrain Pseudomonas putida KT2442 were found to form PHA during theentire fermentation, both during the exponential and during thestationary phase. The use of growth-limiting conditions is therefore notnecessary.

The biopolymer included in the cells need not necessarily be isolated,because the bacterial cells with the biopolymer inclusions therein cansometimes be directly used, as proposed, e.g., in U.S. Pat. No.3,107,172. For most uses, however, isolation and purification of thepolymer will be desirable or necessary. For this purpose, many known perse methods can be used. The bacterial cells can be broken up in manyways which are well known to those skilled in the art, e.g., by usingshear forces (by means of homogenizers, grinders, so-called "Frenchpress" and the like), by carrying out an osmotic shock treatment, byusing sonorous or ultrasonorous vibrations, by enzymatic cell walldecomposition, or by spray drying the cell suspension. Subsequently, thepolymer can be separated from the other components in many known per semanners, including solvent extraction and centrifugation. One suitablemethod of isolation described in the above European patent applicationEP-A-0 274 151 uses isopycnic centrifugation. For isolation on a largerscale it is preferable for the biopolymer to be isolated by convertingthe harvested cells into spheroplasts, breaking these up by a treatmentwith sonorous vibrations, separating and, if desired, washing and dryingthe top layer formed after centrifugation. Preferably, the conversioninto spheroplasts is effected in the presence of sucrose. Centrifugationproceeds satisfactorily if effected for about 30 minutes at 10,000 g.The polymer then forms a white top layer on the supernatant and caneasily be separated. Contaminations can be removed by washing, afterwhich the washed polymer is brought into a suitable dry form, preferablyby freeze drying.

Another suitable procedure for the isolation of the polymer formed isthe procedure described in European patent application EP-A-0 274 151.When compared with the biochemical procedure using a sucrose gradientand different centrifugation steps, extraction of freeze dried cellswith, e.g., chloroform is easier, faster, and conducive to a higheryield of a purer product.

For the uses of the polyesters formed reference is made to the aboveEuropean patent application EP-A-0 274 151, including the possibility ofchemically modifying the resulting biopolymers or crosslinking them withother polymer chains, and the use for the manufacture of products, suchas sutures, films, skin or bone grafts etc.

The invention also provides a process for producing optically activecarboxylic acids or carboxylic acid esters, which comprises hydrolyzinga polyester obtained by the process according to the invention, and, ifdesired, esterifying the resulting monomers. It is often not easy toobtain such optically active compounds, which may have utility, e.g., asintermediates in the manufacture of pharmaceutical products, or forresearch, in an optically pure form by chemical means. If, as a resultof the substrate used, the process produces mixtures of differentmonomers, these can, if desired, be separated in a known per se manner.

The invention is illustrated in and by the following experimentalsection.

(1) Bacterial Strains and Growth Conditions

The strains listed in Table A were used. The growth media used wereE-medium (Vogel and Bonner, J. Biol. Chem. 218, 1956, 97-106) ,E2-medium (Lageveen et al, Appl. Env. Microbiol. 54, 1988, 2924-2932) or0.5xE2.

E-medium has the following composition:

    ______________________________________                                        E-medium has the following composition:                                       trisodium citrate     2.0    g/l                                              MgSO.sub.4.7H.sub.2 O 0.2    g/l                                              NaNH.sub.4 HPO.sub.4.4H.sub.2 O                                                                     3.5    g/l                                              K.sub.2 HPO.sub.4     10     g/l                                              1000 MT               1      ml/l                                             E2-medium has the following composition:                                      NaNH.sub.4 HPO.sub.4.4H.sub.2 O                                                                     3.5    g/l                                              K.sub.2 HPO.sub.4.3H.sub.2 O                                                                        7.5    g/l                                              KH.sub.2 PO.sub.4     3.7    g/l                                              1000 MT               1      ml/l                                             100 mM MgSO.sub.4     10     ml/l                                             0.5 × E2-medium has the following composition:                          NaNH.sub.4 HPO.sub.4.4H.sub.2 O                                                                     3.5    g/l                                              K.sub.2 HPO.sub.4.3H.sub.2 O                                                                        3.8    g/l                                              KH.sub.2 PO.sub.4     1.9    g/l                                              1000 MT               1      ml/l                                             100 mM MgSO.sub.4     10     ml/l                                             1000 MT (in 1 N HCl) has the following composition:                           FeSO.sub.4.7H.sub.2 O 2.78   g/l                                              MnCl.sub.2.4H.sub.2 O 1.98   g/l                                              CoSO.sub.4.7H.sub.2 O 2.81   g/l                                              CaCl.sub.2.2H.sub.2 O 1.47   g/l                                              CuCl.sub.2.2H.sub.2 O 0.17   g/l                                              ZnSO.sub.4.7H.sub.2 O 0.29   g/l                                              ______________________________________                                    

The above media allow growth up to a certain cell density, after whichnitrogen is limiting. The cell density obtainable on the 0.5xE2-mediumis expected to be about 0.7-0.9 mg/ml. at this cell density nitrogen islimiting, and further increase in cell mass is the result of anaccumulation of PHA.

The fatty acids were dissolved in 10% Brij 58 up to final concentrationsof 10 mM (for butyrate, valerate, octanoate, nonanoate, decanoate andundecanoate), 5 mM (for laurate, tridecanoate, myristate,pentadecanoate, oleate, elaidate and γ-linolate) and 2 mM (forpalmirate, heptadecanoate, stearate and erucate) as 10 times moreconcentrated stock solutions, brought to pH 7.0 by adding 1N KOH, andsterilized through a filter (Jenkins and Nunn, J. Bacteriol. 169, 1987,42-52). 3-Hydroxy-butyrate was added up to 0.7%. 1-Octanol and octanal(3% v/v) were added directly to liquid cultures and, in vapor form, tocells growing on solid media. To 1-octanol or octanal containing liquidcultures were added 17% dibutyl phthalate to prevent damage to thecells.

                  TABLE A                                                         ______________________________________                                        strains used                                                                  strain       characteristics                                                                             reference                                          ______________________________________                                        P. oleovorans                                                                 GPo1         OCT           (a)                                                GPo12        OCT.sup.-     Kok, thesis                                        Groningen                                                                     P. putida                                                                     PpG1                       (b)                                                KT2442       TOL.sup.-, Rf.sup.r                                                                         (c)                                                P. aeruginosa                                                                 PA01         prototype     (d)                                                P. fluorescens                                                                             prototype     (e), DSM 50090                                     P. lemonnieri              (e), DSM 50415                                     P. testosteroni                                                                            prototype     (f)                                                ______________________________________                                         abbreviations:                                                                OCT: OCT plasmid;                                                             TOL: TOL plasmid;                                                             Rf.sup.r : rifampicin resistance;                                             DSM: Deutsche Sammlung fur Mikroorganismen und Zellkulturen GmbH,             Braunschweig, FRG.                                                            (a): Schwartz and McCoy, Appl. Microbiol. , 1973, 217-218                     (b): Grund et al, J. Bacteriol. , 1975, 546-556                               (c): Bagdasarian et al, Gene , 1981, 237-247                                  (d): Holloway, Bacter. Rev. 3, 1969, 419-443                                  (e): Stanier et al, J. Gen. Microbiol. , 1966, 159-271                        (f): Marcus and Talalay, J. Biol. Chem. , 1956, 661-674                  

(2) Determination of PHA

For a qualitative analysis of the presence of PHA the cells wereexamined microscopically. For a quantitative analysis the cells werecultured on 0.5xE2 agar plates or in 50 ml 0.5xE2 cultures and testedfor the presence and composition of PHA according to the methoddescribed before by Lageveen et al. The composition of the polymers wasalso determined at isolated polymer obtained from 1 l cultures afterchloroform extraction.

For the polymer test, cells were harvested, lyophilized and then treatedwith 15% sulfuric acid in methanol/chloroform at 100° C for 140 min. toconvert fatty acids into the methyl esters. The methyl esters were thenanalyzed by means of gas-liquid chromatography (GLC). The measurement ofmethyl-3-hydroxy-butyrate was adapted by starting the GLC program at 68°C. After 2 min. at this temperature the column was heated for 20 min. ata rate of 5° C./min. Then the column temperature was increased to 278°C. at a rate of 10° C./min. to remove all the high-molecular components.On the basis of the cell density (Witholt, J. Bacteriol. 109, 1972,350-364) and the peak surface ratios of the monomers and the internalstandard (methyl benzoate) the amount of polymer accumulated by thecells and the composition thereof could be determined.

(3) Formation of PHA by P. oleovorans on paraffin oxidation products

The formation of PHA by the prototype of paraffin oxidizingpseudomonads, namely P. oleovorans GPo1, using 1-octanol, octanal oroctanoate as a substrate, was examined. These carbon sources were addedto final amounts of 3% (vol/vol) or 10 mM (octanoate). Because 1-octanoland octanal were toxic to the cells, the cultures were supplemented with17% dibutyl phthalate. The results are listed in Table B.

When 1-octanol or octanoate was used as a substrate, PHA was actuallyformed. When octanal was used as a substrate, no growth occurred. Theyield of polymer was highest after growth on octanoate. The polymersformed consisted mainly of 3-hydroxy-octanoate units (90%), and the restconsisted of 3-hydroxy-hexanoate units. This corresponds to the resultsobtained using octane.

                  TABLE B                                                         ______________________________________                                        effect of oxidation state of substrate                                        dry cell mass  cellular PHA                                                                             polymer composition                                 substrate                                                                             (mg/ml)    content (%)                                                                              30H-6  30H-8                                    ______________________________________                                        1-octanol                                                                             1.3        3.1        0.10   0.90                                     octanal no growth                                                             octanoate                                                                             1.2        8.7        0.12   0.88                                     ______________________________________                                    

Bacteria of the strain P. oleovorans GPo1 were grown in 50 ml 0.5xE2medium on the substrates referred to and analyzed after 20 hours ofgrowth on PHA. The cellular PHA content is the percentage of polymermass relative to the dry cell mass. By 30H-6 and 30H--8 are meant3-hydroxy-hexanoate and 3-hydroxyoctanoate, respectively.

(4) Formation of PHA by Other Pseudomonads

Not only for bacteria of the strain P. oleovorans GPo1, but also for thestrains P. oleovorans GPo12 (an OCT-derivative of GPo1), P. putida PpG1(a plasmid-less strain isolated from soil), and P. putida KT2442 (arifampicin-resistant strain derived from P. putida mt-2 and stripped ofthe TOL plasmid), which strains do not grow on paraffins, was polymerformation examined. All these strains belong to the group of P. putida,biotype A. The strains were grown on agar plates with minimum medium onoctanol vapor and analyzed microscopically for the presence of PHA. Inall these strains the formation of a reserve material was establishedfrom the intracellular accumulation of polymer granules observed inphase contrast microscopy as transparent (white) dots.

In order to determine the amount and composition of the reservematerial, cells of the plates were collected and analyzed for PHA. Asappears from Table C, the tested strains, except P. putida PpG1,produced PHA, in which 90% consisted of 3-hydroxy-octanoate and 10% of3-hydroxy-hexanoate. The strain P. putida PpG1 also produced PHA, butthis was found to be nearly a homopolymer, because no less than 96%thereof was built up from C₈ and only 4% from C₆ monomers. As comparedwith the P. oleovorans strains GPo1 and GPo12, the P. putida strainsPpG1 and KT2442 proved to accumulate higher intracellular PHA contents.

                  TABLE C                                                         ______________________________________                                        PHA synthesis by other Pseudomonas strains                                    strain                                                                        (%)       Cellular PHA content (%)                                                                       fraction 30H-6                                     ______________________________________                                        GPo1      2-5              9.5 ± 1.0                                       GPo12     2-5              9.4 ± 1.5                                       PpG1      10-20            4.0 ± 0.7                                       KT2442    10-20            9.0 ± 1.1                                       ______________________________________                                    

The strains were grown on 1-octanol on 0.5xE2 plates. The composition isshown by means of the fraction 3-hydroxy-hexanoate monomers in thepolyester, and the rest is formed by 3-hydroxy-octanoate.

(5) Formation of PHA by yet other Pseudomonads

There are different classifications of pseudomonads, such as theclassification according to rRNA homology (de Vos and de Ley, Int. J.Syst. Bacteriol. 33, 1983, 487-509) , according to similarities betweenenzymes involved in the biosynthesis of aromatic amino acids (Byng etal, J. Mol. Evol. 19, 1983, 272-282) , and according to metabolismproperties as described in Bergey's Manual of DeterminativeBacteriology. In all these different classification systems thefluorescent Pseudomonas strains (the species P. putida, P. aeruginosaand P. fluorescens) are members of the same group and have in commonthat they are incapable of forming PHB.

The investigation into the formation of PHA's was continued fordifferent Pseudomonas strains from the P. fluorescens rRNA branch,namely the prototypes P. aeruginosa PAO, P. fluorescens and P.lemonnieri as well as the above-mentioned P. oleovorans and P. putidastrains and further the species P. testosteroni not belonging to thisgroup. All these strains were grown on 10 mM octanoate or on 0.7%3-hydroxy--butyrate in 50 ml 0.5xE2 cultures. The cells were harvested,and PHA was determined at whole cells (Table D).

The P. putida strains were also grown in E2-medium on 30 mM octanoate or0.7% 3-hydroxy-butyrate in 1 l fermenters, followed by isolation of thepolyesters. The composition of the polymers was found to correspond tothe composition determined in the analysis at whole cells. The PHA's ofcells grown on octanoate always consisted of 3-hydroxy-hexanoate,3-hydroxy-octanoate, and small amounts of 3-hydroxy-decanoate. No3-hydroxy fatty acids having an odd number of carbon atoms weredetected. After growth on 3-hydroxy-butyrate, 3-hydroxy fatty acids werenot found in culture samples, nor in the material obtained byprecipitation of a chloroform extract of the whole cells.

Bacteria of the species P. aeruginosa, P. fluorescens and P. lemonnieriwere all found to form PHA after growth on octanoate, while they formedno polymer during growth on 3-hydroxy-butyrate. On the other hand, P.testosteroni, which was incapable of growing on octanoate, was found toform PHB during growth on 3-hydroxy-butyrate or decanoate. During growthon the latter substrate only 13% of the monomers did not consist of3-hydroxy-butyrate. The amounts of PHA formed by the different specieswere widely divergent.

The P. putida and P. oleovorans strains gave 30-50% PHA, P. aeruginosagave about 15%, and P. fluorescens, the prototype of the fluorescentpseudomonads, gave only 1-2% PHA. On the other hand, the species P.lemonnieri belonging to P. fluorescens Biotype IV accumulated PHA up tonearly 40% of its dry cell mass. These percentages, however, only serveas a general indication of the PHA forming capacity of the optionallyfluorescent pseudomonads of the P. fluorescens rRNA branch, because theculture conditions may have an effect on the PHA yield and the PHAsynthesis is not yet optimized by the different strains.

                  TABLE D                                                         ______________________________________                                        PHA formation by different Pseudomonas strains                                sub-       polymer   composition                                              strain strate  content   30H-4 30H-6 30H-8 30H-10                             ______________________________________                                        P. oleo-                                                                      vorans                                                                        GPol   HB      0                                                                     oct     29.7      --    0.07  0.90  0.03                               GPo12  HB      0                                                                     oct     34.1      --    0.06  0.91  0.03                               P. putida                                                                     PpG1   HB      0                                                                     oct     29.4      --    0.03  0.93  0.04                               KT2442 HB      0                                                                     oct     47.1      --    0.08  0.91  0.01                               P. aerug-                                                                     inosa                                                                         PAO    HB      0.1       --    --    --    1.0                                       oct     14.4      --    0.08  0.86  0.07                               P. fluor-                                                                     escens                                                                               HB      0.4       --    --    --    1.0                                       oct     1.6       --    0.14  0.63  0.22                               P. le-                                                                        monneri                                                                       DSM 50415                                                                            HB      0.7       --    --    0.14  0.86                                      oct     38.5      --    0.06  0.91  0.03                               P. testos-                                                                    teroni                                                                               HB      15.4      0.99  --    --    0.01                               oct        no growth                                                          dec         6.0      0.87    0.03  0.09  0.02                                 ______________________________________                                    

The abbreviations used for the substrate are: HB: 3-hydroxy-butyrate;oct: octanoate; and dec: decanoate.

(6) Effect of the Substrate on the PHA Formation

In order to determine the effect of the growth substrate on thecomposition of the polymer formed, P. oleovorans GPo1 was cultured in 50ml cultures containing 0.5xE2-medium supplemented with a fatty acidhaving 4-22 carbon atoms, after which the cells were harvested andtested for the presence of poly-3-hydroxy-alkanoates. The results arelisted in Table E.

The PHA formation was limited when small fatty acids, such as3-hydroxy-butyrate, butyrate and valerate, were used. Despite thelimited production (less than 1.5%) the PHA's formed contained C₈, C₁₀and C₁₂ monomers. Monomers having less than 8 carbon atoms were notobserved.

When carboxylic acids of medium length, such as octanoate, nonanoate,decanoate and undecanoate, were used, a substantial polymer formationtook place (more than 8%). The composition of the polyesterscorresponded to that of the PHA's formed during growth on thecorresponding n-paraffins.

Growth on longer fatty acids also led to PHA synthesis. When laurate,tridecanoate, myristate, pentadecanoate, and palmirate were used as acarbon source, the amount of polymer formed was in the same range as hadbeen found during use of fatty acids of medium length (more than 5%).When longer saturated fatty acids, such-as heptadecanoate and stearate,were used, the bacteria did not grow satisfactorily, and no polymerformation was detected. On the other hand, during use of unsaturatedcarboxylic acids having 18 carbon atoms, growth and polymer formationtook place. This was not the case when erucate, an unsaturated fattyacid having 22 carbon atoms was used.

When a substrate having more than 12 carbon atoms (laurate and higher)was used, the composition of the polyesters was found to be not sosubstrate-dependent anymore: when the substrate had an even number ofcarbon atoms, 3-hydroxy-dodecanoate was always the largest monomer, andwhen the substrate had an odd number of carbon atoms,3-hydroxy-undecanoate was always the largest monomer. The composition ofthe PHA's formed on longer (12 and more carbon atoms) fatty acids havingan even number of carbon atoms showed a fixed ratio for the C₆, C₈, C₁₀and C₁₂ monomers of about 1.3:8.9:4.7:1. The composition of the PHA'sformed on longer (11 and more carbon atoms) fatty acids having an oddnumber of carbon atoms showed a fixed ratio for the C₇, C₉ and C₁₁monomers of about 2.3:3.4:1.

                                      TABLE 3                                     __________________________________________________________________________    PHA formation by P. oleovorans on C.sub.4 -C.sub.22 fatty acids               sub-   polymer-                                                                             polymer composition                                             strate content (%)                                                                          30H-6                                                                             30H-7                                                                             30H-8                                                                             30H-9                                                                             30H-10                                                                            30H-11                                                                            30H-12                                  __________________________________________________________________________    saturated fatty acids                                                         HB     1.2    --  --  0.22                                                                              --  0.57                                                                              --  0.21                                    C4     0.6    --  --  --  --  0.33                                                                              --  0.67                                    C5     0.7    --  --  --  --  0.35                                                                              --  0.65                                    C8     8.7    0.08                                                                              --  0.91                                                                              --  0.01                                                                              --  --                                      C9     9.1    --  0.35                                                                              --  0.65                                                                              --  --  --                                      C10    12.5   0.08                                                                              --  0.75                                                                              --  0.17                                                                              --  --                                      C11    9.8    --  0.28                                                                              --  0.59                                                                              --  0.13                                                                              --                                      C12    6.6    0.06                                                                              --  0.57                                                                              --  0.32                                                                              --  0.05                                    C13    5.4    --  0.32                                                                              --  0.48                                                                              0.05                                                                              0.14                                                                              --                                      C14    10.6   0.07                                                                              --  0.59                                                                              --  0.30                                                                              --  0.04                                    C15    5.3    --  0.32                                                                              --  0.47                                                                              0.08                                                                              0.13                                                                              --                                      C16    3.4    0.08                                                                              --  0.50                                                                              --  0.30                                                                              --  0.12                                    C17 and C18                                                                          no growth                                                              unsaturated fatty acids                                                       ole    7.4    0.09                                                                              --  0.57                                                                              --  0.28                                                                              --  0.06                                    ela    11.2   0.10                                                                              --  0.56                                                                              --  0.27                                                                              --  0.07                                    lin    5.9    0.10                                                                              --  0.57                                                                              --  0.30                                                                              --  0.04                                    eru    no growth                                                              __________________________________________________________________________

The abbreviations used for the substrates are: HB: 3-hydroxy-butyrate;C4: butyrate; C5: valerate; C8 to C18: octanoate to octadecanoate; ole:oleate (cis-9-octadecenoate); ela: elaidate (trans-9-octadecenoate);lin: γ-linolenate (cis-6,9,12-octadecatrienoate); eru: erucate(cis-13-docosenoate).

By "-" is indicated: <0.005.

We claim:
 1. A process for producing poly-3-hydroxyalkanoates composedof repeating units having 6-10 carbon atoms comprising selectingbacteria from the Pseudomonks fluorescens rRNA branch and culturing saidbacteria according to the phylogenetic classification by de Vos and deLey in Int. J. of Syst. Bacteriol. 33, 1983, 487-509, with the exceptionof Pseudomonas oleovorans, under aerobic conditions in a nutrient mediumcomprising an excess of a carbon source and a limiting quantity of atleast one of the other nutrients essential for growth, said carbonsource comprising at least one assimilable acyclic aliphatic oxidatedhydrocarbon compound having 6-18 carbon atoms.
 2. A process according toclaim 1, in which bacteria are used selected from the group consistingofPseudomonas fluorescens Pseudomonas lemonnieri Pseudomonas putidabiotype A (except P. oleovorans) Pseudomonas aeruginosa.
 3. A processaccording to claim 1, in which the assimilable acyclic aliphatichydrocarbon compound used is a saturated or unsaturated C₆₋₁₈ paraffinoxidation product.
 4. A process according to claim 3, in which aparaffin carboxylic acid having 6-18 carbon atoms is used.
 5. A processaccording to claim 4, in which a paraffin carboxylic acid having 12-16carbon atoms is used.
 6. A process according to claim 1, in which theaerobic cultivation is carried out with nitrogen, phosphorus, sulfur ormagnesium limitation.
 7. A process according to claim 1, in which theaerobic cultivation is carried out at pH 5-9, at a temperature below 37°C., and with adequate agitation at a dissolved oxygen tension above 30%,saturation with air.
 8. A process according to claim 1, in which theaerobic cultivation with nutrient limitation is preceded by anexponential growth phase without nutrient limitations until a celldensity of at least 2 g/l is reached.
 9. The process of claim 1, furthercomprising recovering the biopolymer formed from the cells.
 10. Aprocess according to claim 9, in which the biopolymer containing cellsformed in the stationary phase with nutrient limitation are harvestedbefore a significant decrease of the biopolymer content of the cells hastaken place.
 11. A process according to claim 9, in which the biopolymeris isolated by converting the harvested cells into spheroplasts,breaking these up by a treatment with sonorous vibrations, separatingand, if desired, washing and drying the top layer formed aftercentrifugation, or isolating the biopolymer by solvent extraction.
 12. Aprocess according to claim 9, in which the resulting biopolymer isisolated and is then chemically modified by at least one furtherchemical reaction.
 13. A process for producing optically activecarboxylic acids or carboxylic acid esters, which comprises the steps ofproducing poly-3-hydroxyalkanoates composed of repeating units having6-10 carbon atoms by the process according to claim 1, hydrolyzing thepoly-3-hydroxyalkanoates obtained and, if desired, esterifying theresulting monomers.
 14. The process of claim 6, wherein the aerobiccultivation is carried out with nitrogen limitation.
 15. The process ofclaim 7, wherein the aerobic cultivation is carried out at about pH 7.16. The process of claim 7, wherein the aerobic cultivation is carriedout at a temperature of about 30° C.
 17. The process of claim 7, whereinthe aerobic cultivation is carried out at a dissolved oxygen tension ofabout 50% or more saturation with air.